Quantitative PCR used to assess HIV-1 integration and 2-LTR circle formation in human macrophages, peripheral blood lymphocytes and a CD4+ cell line

Brian Friedrich, Guangyu Li, Natallia Dziuba, Monique Ferguson

Research output: Contribution to journalArticle

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Abstract

Background. Integration is an intermediate step in the HIV life cycle and is defined as the insertion of HIV-1 proviral DNA into the host chromosome. If integration does not occur when HIV-1 cDNA enters the nucleus, it circularizes upon itself and forms a 2-LTR circle. Monitoring the level of integrated HIV-1 cDNA in different primary cell subsets is very important, particularly regarding the effect of HAART in HIV-1 infected individuals. Because of limitations of prior HIV-1 integration assays, there is limited data on the level of integration and 2-LTR circle formation in primary cell subsets, particularly in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL). Results. In this study, we utilized a well-defined, sensitive two-step quantitative real-time PCR method to detect HIV-1 integration as well as conventional real-time PCR to detect 2-LTR circle formation in human macrophages and PBL isolated from six different healthy donors, as well as U373 CD4 + cells by infecting with HIV-1SX(R5) or dual-tropic isolate HIV-189.6(R5/X4) virus strains. We used the FDA-approved integrase inhibitor, raltegravir, to determine quantitative differences of integrated HIV viral cDNA in HIV-1 infected cells with and without raltegravir treatment. Our results show that integration and 2-LTR circle formation can be assessed in primary macrophages, PBL, and a CD4+ cell line by this method. Specifically, our results demonstrate that this two-step real-time PCR method can distinguish between HIV-1 integrated viral cDNA and non-integrated nuclear HIV-1 2-LTR circles caused by impaired integration with raltegravir-treatment. This further confirms that only integrated HIV-1 cDNA can be specifically amplified and quantified by two-step PCR without non-specifically detecting non-integrated viral cDNA. Conclusion. These results consistently demonstrate that the well-established real-time PCR assays used are robust, sensitive and quantitative for the detection of HIV-1 integration and 2-LTR circle formation in physiologically relevant human macrophages and PBL using lab-adapted virus strains, instead of pseudovirus. With two-step real-time PCR, we show that unintegrated, nuclear HIV-1 cDNA is not detected in raltegravir-treated cells, while specific for only integrated HIV-1 cDNA in non-treated cells. These methods could be applied as a useful tool in further monitoring specific therapy in HIV-1 infected individuals.

Original languageEnglish (US)
Article number354
JournalVirology Journal
Volume7
DOIs
StatePublished - 2010

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HIV-2
HIV-1
Macrophages
Lymphocytes
Cell Line
Polymerase Chain Reaction
Complementary DNA
Real-Time Polymerase Chain Reaction
HIV
Integrase Inhibitors
HIV Long Terminal Repeat
Viruses
Highly Active Antiretroviral Therapy
Life Cycle Stages

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Quantitative PCR used to assess HIV-1 integration and 2-LTR circle formation in human macrophages, peripheral blood lymphocytes and a CD4+ cell line. / Friedrich, Brian; Li, Guangyu; Dziuba, Natallia; Ferguson, Monique.

In: Virology Journal, Vol. 7, 354, 2010.

Research output: Contribution to journalArticle

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abstract = "Background. Integration is an intermediate step in the HIV life cycle and is defined as the insertion of HIV-1 proviral DNA into the host chromosome. If integration does not occur when HIV-1 cDNA enters the nucleus, it circularizes upon itself and forms a 2-LTR circle. Monitoring the level of integrated HIV-1 cDNA in different primary cell subsets is very important, particularly regarding the effect of HAART in HIV-1 infected individuals. Because of limitations of prior HIV-1 integration assays, there is limited data on the level of integration and 2-LTR circle formation in primary cell subsets, particularly in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL). Results. In this study, we utilized a well-defined, sensitive two-step quantitative real-time PCR method to detect HIV-1 integration as well as conventional real-time PCR to detect 2-LTR circle formation in human macrophages and PBL isolated from six different healthy donors, as well as U373 CD4 + cells by infecting with HIV-1SX(R5) or dual-tropic isolate HIV-189.6(R5/X4) virus strains. We used the FDA-approved integrase inhibitor, raltegravir, to determine quantitative differences of integrated HIV viral cDNA in HIV-1 infected cells with and without raltegravir treatment. Our results show that integration and 2-LTR circle formation can be assessed in primary macrophages, PBL, and a CD4+ cell line by this method. Specifically, our results demonstrate that this two-step real-time PCR method can distinguish between HIV-1 integrated viral cDNA and non-integrated nuclear HIV-1 2-LTR circles caused by impaired integration with raltegravir-treatment. This further confirms that only integrated HIV-1 cDNA can be specifically amplified and quantified by two-step PCR without non-specifically detecting non-integrated viral cDNA. Conclusion. These results consistently demonstrate that the well-established real-time PCR assays used are robust, sensitive and quantitative for the detection of HIV-1 integration and 2-LTR circle formation in physiologically relevant human macrophages and PBL using lab-adapted virus strains, instead of pseudovirus. With two-step real-time PCR, we show that unintegrated, nuclear HIV-1 cDNA is not detected in raltegravir-treated cells, while specific for only integrated HIV-1 cDNA in non-treated cells. These methods could be applied as a useful tool in further monitoring specific therapy in HIV-1 infected individuals.",
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PY - 2010

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N2 - Background. Integration is an intermediate step in the HIV life cycle and is defined as the insertion of HIV-1 proviral DNA into the host chromosome. If integration does not occur when HIV-1 cDNA enters the nucleus, it circularizes upon itself and forms a 2-LTR circle. Monitoring the level of integrated HIV-1 cDNA in different primary cell subsets is very important, particularly regarding the effect of HAART in HIV-1 infected individuals. Because of limitations of prior HIV-1 integration assays, there is limited data on the level of integration and 2-LTR circle formation in primary cell subsets, particularly in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL). Results. In this study, we utilized a well-defined, sensitive two-step quantitative real-time PCR method to detect HIV-1 integration as well as conventional real-time PCR to detect 2-LTR circle formation in human macrophages and PBL isolated from six different healthy donors, as well as U373 CD4 + cells by infecting with HIV-1SX(R5) or dual-tropic isolate HIV-189.6(R5/X4) virus strains. We used the FDA-approved integrase inhibitor, raltegravir, to determine quantitative differences of integrated HIV viral cDNA in HIV-1 infected cells with and without raltegravir treatment. Our results show that integration and 2-LTR circle formation can be assessed in primary macrophages, PBL, and a CD4+ cell line by this method. Specifically, our results demonstrate that this two-step real-time PCR method can distinguish between HIV-1 integrated viral cDNA and non-integrated nuclear HIV-1 2-LTR circles caused by impaired integration with raltegravir-treatment. This further confirms that only integrated HIV-1 cDNA can be specifically amplified and quantified by two-step PCR without non-specifically detecting non-integrated viral cDNA. Conclusion. These results consistently demonstrate that the well-established real-time PCR assays used are robust, sensitive and quantitative for the detection of HIV-1 integration and 2-LTR circle formation in physiologically relevant human macrophages and PBL using lab-adapted virus strains, instead of pseudovirus. With two-step real-time PCR, we show that unintegrated, nuclear HIV-1 cDNA is not detected in raltegravir-treated cells, while specific for only integrated HIV-1 cDNA in non-treated cells. These methods could be applied as a useful tool in further monitoring specific therapy in HIV-1 infected individuals.

AB - Background. Integration is an intermediate step in the HIV life cycle and is defined as the insertion of HIV-1 proviral DNA into the host chromosome. If integration does not occur when HIV-1 cDNA enters the nucleus, it circularizes upon itself and forms a 2-LTR circle. Monitoring the level of integrated HIV-1 cDNA in different primary cell subsets is very important, particularly regarding the effect of HAART in HIV-1 infected individuals. Because of limitations of prior HIV-1 integration assays, there is limited data on the level of integration and 2-LTR circle formation in primary cell subsets, particularly in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL). Results. In this study, we utilized a well-defined, sensitive two-step quantitative real-time PCR method to detect HIV-1 integration as well as conventional real-time PCR to detect 2-LTR circle formation in human macrophages and PBL isolated from six different healthy donors, as well as U373 CD4 + cells by infecting with HIV-1SX(R5) or dual-tropic isolate HIV-189.6(R5/X4) virus strains. We used the FDA-approved integrase inhibitor, raltegravir, to determine quantitative differences of integrated HIV viral cDNA in HIV-1 infected cells with and without raltegravir treatment. Our results show that integration and 2-LTR circle formation can be assessed in primary macrophages, PBL, and a CD4+ cell line by this method. Specifically, our results demonstrate that this two-step real-time PCR method can distinguish between HIV-1 integrated viral cDNA and non-integrated nuclear HIV-1 2-LTR circles caused by impaired integration with raltegravir-treatment. This further confirms that only integrated HIV-1 cDNA can be specifically amplified and quantified by two-step PCR without non-specifically detecting non-integrated viral cDNA. Conclusion. These results consistently demonstrate that the well-established real-time PCR assays used are robust, sensitive and quantitative for the detection of HIV-1 integration and 2-LTR circle formation in physiologically relevant human macrophages and PBL using lab-adapted virus strains, instead of pseudovirus. With two-step real-time PCR, we show that unintegrated, nuclear HIV-1 cDNA is not detected in raltegravir-treated cells, while specific for only integrated HIV-1 cDNA in non-treated cells. These methods could be applied as a useful tool in further monitoring specific therapy in HIV-1 infected individuals.

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